PROJECT SUMMARY Loss of B cell tolerance and production of auto-antibodies (Ab) by plasma cells (PCs) play a major role in Systemic Lupus Erythematosus (SLE) pathology. Though Ab-dependent pathogenesis can be partially controlled with immunosuppression, there is no cure for Ab-mediated disorders. One of the main limitations when developing therapeutic strategies aimed to prevent Ab-dependent pathology is the lack of precise understanding of how auto-reactive PCs are generated and maintained. In this regard, T follicular helper (Tfh) cells, a subset of CD4+ T cells that provides help to B cells, are considered to play a critical role in promoting auto-reactive PCs. As such, the expansion of self-reactive Tfh cells correlates with auto-Ab production and disease severity in murine and human lupus. Similar to Tfh cells, Double-negative (DN) CD3+CD4-CD8- T cells, a particular population of T cells that characteristically lack CD4 and CD8 expression, are also abnormally expanded in lupus. Importantly, the frequency of DN T cells also correlates with disease activity and auto-Ab production. Thus, it is generally believed that these cells play a role in autoimmune disease pathogenesis. Despite their putative role in disease development, however, we do not know what signals control DN T cell formation, and their exact origin and pathogenic function remain largely elusive. Furthermore, the mechanisms that regulate DN T cell homeostasis are entirely unknown, and there are currently no therapies to selectively deplete DN T cells in vivo. The main goal of this proposal is to define the cellular and molecular mechanisms that control DN T cell development and function in lupus-prone mice and SLE patients. In this regard, our preliminary data demonstrate that DN T cells and Tfh cells share essential phenotypic, transcriptional, and developmental requirements. The central hypothesis that will be tested in this proposal is that Tfh cells are the precursors of DN T cells and that, similar to Tfh cells, DN T cells are efficient B cell helpers. Importantly, our preliminary data also suggest that DN T cells are more plastic than Tfh cells, which allows them to acquire a ?hybrid? Tfh/Th17 signature that we believe is critical for supporting auto-reactive PC responses. In Aim 1, we will test the hypothesis that Tfh cells are precursors of DN T cells and examine the capacity of DN T cells to help self-reactive B cell responses. In Aim 2.1, we will test the hypothesis that IL-17 production by DN T cells is critical for supporting extrafollicular PC responses and Ab-mediated pathology. In Aim 2.2, we will determine the molecular mechanisms controlling the acquisition of a ?hybrid? Tfh/Th17 signature. Finally, in Aim 3, we will develop a new synergistic IL-2-based immunotherapy aimed to selectively target Tfh and prevent the differentiation of IL-17+CXCR5+DN T cells by combining ?ultra-low? doses of rIL-2 with STAT3-signaling blockade. We believe that our studies will provide a new paradigm for how DN T cells are generated, will reveal new pathways implicated in autoimmune disease pathogenesis, and will be crucial for designing new therapeutic interventions to target Tfh and DN T cells and prevent Ab-mediated pathology.